Microwave method for preparing radiolabelled gallium complexes

ABSTRACT

The present invention relates to a method of producing radiolabelled gallium complexes that could be used as diagnostic agents, e.g. for positron emission tomography (PET) imaging.

This application is a filing under 35 U.S.C. 371 of internationalapplication number PCT/GB2004/001550, filed Apr. 8, 2004, which claimspriority to application number 0308408.4 filed Apr. 11, 2003, in GreatBritain the entire disclosure of which is hereby incorporated byreference.

The present invention relates to a method of producing radiolabelledgallium complexes. The complexes could be used as diagnostic agents,e.g. for positron emission tomography (PET) imaging.

PET imaging is a tomographic nuclear imaging technique that usesradioactive tracer molecules that emit positrons. When a positron meetsan electron, the both are annihilated and the result is a release ofenergy in form of gamma rays, which are detected by the PET scanner. Byemploying natural substances that are used by the body as tracermolecules, PET does not only provide information about structures in thebody but also information about the physiological function of the bodyor certain areas therein. A common tracer molecule is for instance2-fluoro-2-deoxy-D-glucose (FDG), which is similar to naturallyoccurring glucose, with the addition of a ¹⁸F-atom. Gamma radiationproduced from said positron-emitting fluorine is detected by the PETscanner and shows the metabolism of FDG in certain areas or tissues ofthe body, e.g. in the brain or the heart. The choice of tracer moleculedepends on what is being scanned. Generally, a tracer is chosen thatwill accumulate in the area of interest, or be selectively taken up by acertain type of tissue, e.g. cancer cells. Scanning consists of either adynamic series or a static image obtained after an interval during whichthe radioactive tracer molecule enters the biochemical process ofinterest. The scanner detects the spatial and temporal distribution ofthe tracer molecule. PET also is a quantitative imaging method allowingthe measurement of regional concentrations of the radioactive tracermolecule.

Commonly used radionuclides in PET tracers are ¹¹C, ¹⁸F, ¹⁵O ¹³N or⁷⁶Br. Recently, new PET tracers were produced that are based onradiolabelled metal complexes comprising a bifunctional chelating agentand a radiometal Bifunctional chelating agents are chelating agents thatcoordinate to a metal ion and are linked to a targeting vector that willbind to a target site in the patient's body. Such a targeting vector maybe a peptide that binds to a certain receptor, probably associated witha certain area in the body or with a certain disease. A targeting vectormay also be an oligonucleotide specific for e.g. an activated oncogeneand thus aimed for tumour localisation. The advantage of such complexesis that the bifunctional chelating agents may be labelled with a varietyof radiometals like, for instance, ⁶⁸Ga, ²¹³Bi or ⁸⁶Y. In this way,radiolabelled complexes with special properties may be “tailored” forcertain applications.

⁶⁸Ga is of special interest for the production of Ga-radiolabelled metalcomplexes used as tracer molecules in PET imaging. ⁶⁸Ga is obtained froma ⁶⁸Ge/⁶⁸Ga generator, which means that no cyclotron is required. ⁶⁸Gadecays to 89% by positron emission of 2.92 MeV and its 68 min half lifeis sufficient to follow many biochemical processes in vivo withoutunnecessary radiation. With its oxidation state of +III, ⁶⁸Ga formsstable complexes with various types of chelating agents and ⁶⁸Ga tracershave been used for brain, renal, bone, blood pool, lung and tumourimaging.

J. Schumacher et al., Cancer Res. 61, 2001, 3712-3717 describe thesynthesis of⁶⁸Ga—N,N′[2-hydroxy-5-(ethylene-β-carboxy)benzyl]ethylenediamine-N,N′-diaceticacid (⁶⁸Ga-HBED-CC). ⁶⁸Ga obtained from a ⁶⁸Ge/⁶⁸Ga generator and Ga³⁺carrier are reacted with the chelating agent HBED-CC in acetate bufferfor 15 min at 95° C. Uncomplexed ⁶⁸Ga is separated from the complexusing a cation exchange column. The overall preparation is reported totake 70 min. A disadvantage of this method is that the overallpreparation time of the radiolabelled complex is very long. Due to theaddition of “cold” Ga³⁺ carrier, the specific activity of the reactionis low. Moreover, the radiolabelled complex had to be purified after thecomplex formation reaction.

WO-A-99/56791 discloses the reaction of ⁶⁸GaCl₃ obtained from a⁶⁸Ge/⁶⁸Ga generator with the tetradentate amine trithiolate chelatingagent tris(2-mercaptobenzyl)amine (S₃N). The complex formation iscarried out at room temperature for 10 min. A disadvantage of the methoddescribed is that the radiolabelled complex had to be purified by liquidchromatography before it could be used for in vivo studies. A furtherdisadvantage of the method is the relatively long reaction time.

Ö. Ugur et al., Nucl. Med. Biol. 29, 2002, 147-157 describe thesynthesis of the ⁶⁸Ga labelled somatostatin analogueDOTA-DPhe¹-Tyr³-octreotide (DOTATOC). The compound is prepared byreacting ⁶⁸GaCl₃ obtained from a ⁶⁸Ge/⁶⁸Ga generator with the chelatingagent DOTATOC for 15 min at 100° C. A disadvantage of this method isthat the reaction mixture had to be heated at relatively hightemperatures. The DOTA chelating agent was functionalised with a peptidetargeting vector and peptides and proteins are substances, which areknown to be sensitive to heat. Thus, with the method described there isa risk that heat sensitive targeting vectors are destroyed duringcomplex formation. A further disadvantage is that the complex had to bepurified by HPLC before it could be used for animal studies.

U.S. Pat. No. 5,070,346 discloses ⁶⁸Ga-labelled complexes of thechelating agent tetraethylcyclohexyl-bis-aminoethanethiol (BAT-TECH).The complexes are synthesised by reacting ⁶⁸GaCl₃ obtained from a⁶⁸Ge/⁶⁸Ga generator with BAT-TECH at 75° C. for 15 min and subsequentfiltration. The preparation of the complex was accomplished in 40 min.Due to the high reaction temperature; this method would not be suitablefor bifunctional chelating agents comprising a heat sensitive targetingvector, for instance a peptide or a protein. A further disadvantage isthe long reaction time of the complex formation reaction.

In view of the relatively short half-life of ⁶⁸Ga there is a need for afast method for the synthesis of ⁶⁸Ga-labelled complexes, which could beused as tracer molecules for PET imaging.

It has now been found that the use of microwave activation substantiallyimproves the efficiency and reproducibility of the ⁶⁸Ga-chelating agentcomplex formation. Due to microwave activation, chemical reaction timescould be shortened substantially; i.e. the reaction is completed within2 min and less. This is a clear improvement as a 10 minutes shortage ofthe reaction time saves about 10% of the ⁶⁸Ga activity. Furthermore,microwave activation also leads to fewer side reactions and to anincreased radiochemical yield, which is due to increased selectivity.Solutions of ⁶⁶Ga³⁺, ⁶⁷Ga³⁺ and ⁶⁸Ga³⁺ radioisotopes, which have beenobtained by cyclotron production or from a generator contain so-calledpseudo carriers, i.e. other metal cations like for instance Fe³⁺, Al³⁺,Cu²⁺, Zn²⁺ and In³⁺. As these pseudo carriers compete with Ga³⁺ in thecomplex formation reaction, it is important to increase the selectivityof the radiolabelling reaction. Hence, microwave activation has apositive effect on radiolabelling with all Ga-radioisotopes, namely with⁶⁶Ga, ⁶⁷Ga and ⁶⁸Ga.

Microwave activation has been used in nucleophilic aromaticradiofluorations with ¹⁸F and it was found that comparable or betteryields than those reported for thermal treatments were obtained inshorter reaction times (S. Stone-Elander et al., Appl. Rad. Isotopes44(5), 1993, 889-893). However, the use of microwave activation inGa-radiolabelling reactions has not been described yet.

The invention thus provides a method of producing a radiolabelledgallium complex by reacting a Ga³⁺ radioisotope with a chelating agentcharacterised in that the reaction is carried out using microwaveactivation.

Suitable Ga³⁺ radioisotopes according to the invention are ⁶⁶Ga³⁺,⁶⁷Ga³⁺ and ⁶⁸Ga³⁺, preferably ⁶⁶Ga³⁺ and ⁶⁸Ga³⁺ and particularlypreferably ⁶⁸Ga³⁺. ⁶⁶Ga³⁺ and ⁶⁸Ga³⁺ are particularly suitable for theproduction of radiolabelled complexes useful in PET imaging whereas⁶⁷Ga³⁺ is particularly suitable for the production of radiolabelledcomplexes useful in single photon emission computerised tomography(SPECT).

⁶⁶Ga³⁺ is obtainable by cyclotron production by irradiation of elementalzinc targets. To minimise the amounts of ⁶⁷Ga production, the targetthickness is preferably maintained such that the degraded proton energyis above 8 MeV, and irradiation time is kept short, e.g. <4 hrs. Thechemical separation may be achieved using solvent-solvent extractiontechniques using isopropyl ether and HCl as described in L. C. Brown,Int. J. Appl. Radiat. Isot. 22, 1971, 710-713. ⁶⁶Ga has a relativelylong half-life of 9.5 h and the most abundant positron emitted has auniquely high energy of 4.2 MeV.

⁶⁷Ga³⁺ is obtainable by cyclotron production and ⁶⁷GaCl₃ obtained bycyclotron production is a commercially available compound. The half-lifeof ⁶⁷Ga is 78 h.

⁶⁸Ga is obtainable from a ⁶⁸Ge/⁶⁸Ga generator. Such generators are knownin the art and for instance described by C. Loc'h et al, J. Nucl. Med.21, 1980, 171-173. Generally, ⁶⁸Ge is loaded onto a column consisting ofan organic resin or an inorganic metal oxide like tin dioxide, aluminiumdioxide or titanium dioxide. ⁶⁸Ga is eluted from the column with aqueousHCl, yielding ⁶⁸GaCl₃. ⁶⁸Ga³⁺ is particularly preferred in the methodaccording to the invention as its production does not require acyclotron and its 68 min half-life is sufficient to follow manybiochemical processes in vivo by PET imaging without long radiation.

Preferred chelating agents for use in the method of the invention arethose which present the Ga³⁺ radioisotopes in a physiologicallytolerable form. Further preferred chelating agents are those that formcomplexes with Ga³⁺ radioisotopes that are stable for the time neededfor diagnostic investigations using the radiolabelled complexes.

Suitable chelating agents are, for instance, polyaminopolyacid chelatingagents like DTPA, EDTA, DTPA-BMA, DOA3, DOTA, HP-DOA3, TMT or DPDP.Those chelating agents are well known for radiopharmaceuticals andradiodiagnosticals. Their use and synthesis are described in, forexample, U.S. Pat. No. 4,647,447, U.S. Pat. No. 5,362,475, U.S. Pat. No.5,534,241, U.S. Pat. No. 5,358,704, U.S. Pat. No. 5,198,208, U.S. Pat.No. 4,963,344, EP-A-230893, EP-A-130934, EP-A-606683, EP-A-438206,EP-A-434345, WO-A-97/00087, WO-A-96/40274, WO-A-96/30377, WO-A-96/28420,WO-A-96/16678, WO-A-96/11023, WO-A-95/32741, WO-A-95/27705,WO-A-95/26754, WO-A-95/28967, WO-A-95/28392, WO-A-95/24225,WO-A-95/17920, WO-A-95/15319, WO-A-95/09848, WO-A-94/27644,WO-A-94/22368, WO-A-94/08624, WO-A-93/16375, WO-A-93/06868,WO-A-92/11232, WO-A-92/09884, WO-A-92/08707, WO-A-91/15467,WO-A-91/10669, WO-A-91/10645, WO-A-91/07191, WO-A-91/05762,WO-A-90/12050, WO-A-90/03804, WO-A-89/00052, WO-A-89/00557,WO-A-88/01178, WO-A-86/02841 and WO-A-86/02005.

Suitable chelating agents include macrocyclic chelating agents e.g.porphyrin-like molecules and pentaaza-macrocycles as described by Zhanget al., Inorg. Chem. 37(5), 1998, 956-963, phthalocyanines, crownethers, e.g. nitrogen crown ethers such as the sepulchrates, cryptatesetc., hemin (protoporphyrin IX chloride), heme and chelating agentshaving a square-planar symmetry.

Macrocyclic chelating agents are preferably used in the method of theinvention. In a preferred embodiment, these macrocyclic chelating agentscomprise at least one hard donor atom such as oxygen and/or nitrogenlike in polyaza- and polyoxomacrocycles. Preferred examples ofpolyazamacrocyclic chelating agents include DOTA, TRITA, TETA and HETAwith DOTA being particularly preferred.

Particularly preferred macrocyclic chelating agents comprise functionalgroups such as carboxyl groups or amine groups which are not essentialfor coordinating to Ga³⁺ and thus may be used to couple other molecules,e.g. targeting vectors, to the chelating agent. Examples of suchmacrocyclic chelating agents comprising functional groups are DOTA,TRITA or HETA.

In a further preferred embodiment, bifunctional chelating agents areused in the method according to the invention. “Bifunctional chelatingagent” in the context of the invention means chelating agents that arelinked to a targeting vector. Suitable targeting vectors forbifunctional chelating agents useful in the method according to theinvention are chemical or biological moieties, which bind to targetsites in a patient's body, when the radiolabelled gallium complexescomprising said targeting vectors have been administered to thepatient's body. Suitable targeting vectors for bifunctional chelatingagents useful in the method according to the invention are proteins,glycoproteins, lipoproteins, polypeptides like antibodies or antibodyfragments, glycopolypeptides, lipopolypeptides, peptides, like RGDbinding peptides, glycopeptides, lipopeptides, carbohydrates, nucleicacids e.g. DNA, RNA, oligonucleotides like antisense oligonucleotides ora part, a fragment, a derivative or a complex of the aforesaidcompounds, or any other chemical compound of interest like relativelysmall organic molecules, particularly small organic molecules of lessthan 2000 Da.

In a particularly preferred embodiment, macrocyclic bifunctionalchelating agents are used in the method according to the invention.Preferred macrocyclic bifunctional chelating agents comprise DOTA, TRITAor HETA linked to a targeting vector, preferably to a targeting vectorselected from the group consisting of proteins, glycoproteins,lipoproteins, polypeptides, glycopolypeptides, lipopolypeptides,peptides, glycopeptides, lipopeptides carbohydrates, nucleic acids,oligonucleotides or a part, a fragment, a derivative or a complex of theaforesaid compounds and small organic molecules; particularly preferablyto a targeting vector selected from the group consisting of peptides andoligonucleotides.

The targeting vector can be linked to the chelating agent via a linkergroup or via a spacer molecule. Examples of linker groups aredisulfides, ester or amides, examples of spacer molecules are chain-likemolecules, e.g. lysin or hexylamine or short peptide-based spacers. In apreferred embodiment, the linkage between the targeting vector and thechelating agent part of radiolabelled gallium complex is as such thatthe targeting vector can interact with its target in the body withoutbeing blocked or hindered by the presence of the radiolabelled galliumcomplex.

Microwave activation according to the invention is suitably carried outby using a microwave oven, preferably by using a monomodal microwaveoven as. Suitably microwave activation is carried out at 80 to 120 W,preferably at 90 to 110 W, particularly preferably at about 100 W.Suitable microwave activation times range from 20 s to 2 min, preferablyfrom 30 s to 90 s, particularly preferably from 45 s to 60 s.

A temperature control of the reaction is advisable when temperaturesensitive chelating agents, like for instance bifunctional chelatingagents comprising peptides or proteins as targeting vectors, areemployed in the method according to the invention. Duration of themicrowave activation should be adjusted in such a way, that thetemperature of the reaction mixture does not lead to the decompositionof the chelating agent and/or the targeting vector. If chelating agentsused in the method according to the invention comprise peptides orproteins, higher temperatures applied for a shorter time are generallymore favourable than lower temperatures applied for a longer timeperiod.

Microwave activation can be carried out continuously or in severalmicrowave activation cycles during the course of the reaction.

In a preferred embodiment, the invention provides a method of producinga ⁶⁸Ga radiolabelled PET imaging tracer by reacting ⁶⁸Ga³⁺ with amacrocyclic bifunctional chelating agent comprising hard donor atoms,characterised in that the reaction is carried out using microwaveactivation.

In a particularly preferred embodiment of the method described in thelast preceding paragraph, the microwave activation is carried out from30 s to 90 s at 90 to 110 W.

If ⁶⁸Ga³⁺ is used in the method according to the invention, the ⁶⁸Ga³⁺is preferably obtained by contacting the eluate form a ⁶⁸Ge/⁶⁸Gagenerator with an anion exchanger and eluting ⁶⁸Ga³⁺ from said anionexchanger. In a preferred embodiment, the anion exchanger is an anionexchanger comprising HCO₃ ⁻ as counterions.

The use of anion exchangers to treat ⁶⁸Ga eluate obtained from a⁶⁸Ge/⁶⁸Ga generator is described by J. Schuhmacher et al. Int. J. appl.Radiat. Isotopes 32, 1981, 31-36. A Bio-Rad AG 1×8 anion exchanger wasused for treating the 4.5 N HCl ⁶⁸Ga eluate obtained from a ⁶⁸Ge/⁶⁸Gagenerator in order to decrease the amount of ⁶⁸Ge present in the eluate.

It has now been found that the use of anion exchangers comprising HCO₃ ⁻as counterions is particularly suitable for the purification andconcentration of the generator eluate. Not only the amount of ⁶⁸Gepresent in the eluate could be reduced but also the amount of so-calledpseudo carriers, i.e. other metal cations like Fe³⁺, Al³⁺, Cu²⁺, Zn²⁺and In³⁺, that are eluted together with the ⁶⁸Ga³⁺ from the generator.As these pseudo carriers compete with ⁶⁸Ga³⁺ in the subsequent complexformation reaction, it is especially favourable to reduce the amount ofthose cations as much as possible before the labelling reaction. Afurther advantage of the anion-exchange purification step is that theconcentration of ⁶⁸Ga³⁺, which is in the picomolar to nanomolar rangeafter the elution, can be increased up to a nanomolar to micromolarlevel. Hence, it is possible to reduce the amount of chelating agent ina subsequent complex formation reaction, which considerably increasesthe specific radioactivity. This result is important for the productionof ⁶⁸Ga-radiolabelled PET tracers that comprise a bifunctional chelatingagent; i.e. a chelating agent linked to a targeting vector, as theincrease in specific radioactivity enables the reduction in amount ofsuch tracers when used in a patient.

Hence, another preferred embodiment of the method according to theinvention is a method of producing a ⁶⁸Ga-radiolabelled complex byreacting ⁶⁸Ga³⁺ with a chelating agent using microwave activation,wherein the ⁶⁸Ga³⁺ is obtained by contacting the eluate form a ⁶⁸Ge/⁶⁸Gagenerator with an anion exchanger, preferably with an anion exchangercomprising HCO₃ ⁻ as counterions, and eluting ⁶⁸Ga³⁺ from said anionexchanger.

⁶⁸Ge/⁶⁸Ga generators are known in the art, see for instance C. Loc'h etal, J. Nucl. Med. 21, 1980, 171-173 or J. Schuhmacher et al. Int. J.appl. Radiat. Isotopes 32, 1981, 31-36. ⁶⁸Ge may be obtained bycyclotron production by irradiation of, for instance Ga₂(SO₄)₃ with 20MeV protons. It is also commercially available, e.g. as ⁶⁸Ge in 0.5 MHCl. Generally, ⁶⁸Ge is loaded onto a column consisting of organic resinor an inorganic metal oxide like tin dioxide, aluminium dioxide ortitanium dioxide. ⁶⁸Ga is eluted from the column with aqueous HClyielding ⁶⁸GaCl₃.

Suitable columns for ⁶⁸Ge/⁶⁸Ga generators consist of inorganic oxideslike aluminium dioxide, titanium dioxide or tin dioxide or organicresins like resins comprising phenolic hydroxyl groups (U.S. Pat. No.4,264,468) or pyrogallol (J. Schuhmacher et al., Int. J. appl. Radiat.Isotopes 32, 1981, 31-36). In a preferred embodiment, a ⁶⁸Ge/⁶⁸Gagenerator comprising a column comprising titanium dioxide is used in themethod according to the invention.

The concentration of the aqueous HCl used to elute the ⁶⁸Ga from the⁶⁸Ge/⁶⁸Ga generator column depends on the column material. Suitably 0.05to 5 M HCl is used for elution of ⁶⁸Ga. In a preferred embodiment, theeluate is obtained from a ⁶⁸Ge/⁶⁸Ga generator comprising a columncomprising titanium dioxide and ⁶⁸Ga is eluted using 0.05 to 0.1 M HCl,preferably about 0.1 M HCl.

In a preferred embodiment of the method according to the invention, astrong anion exchanger comprising HCO₃ ⁻ as counterions, preferably astrong anion exchanger comprising HCO₃ ⁻ as counterions, is used. In afurther preferred embodiment, this anion exchanger comprises quaternaryamine functional groups. In another further preferred embodiment, thisanion exchanger is a strong anion exchange resin based onpolystyrene-divinylbenzene. In a particularly preferred embodiment, theanion exchanger used in the method according to the invention is astrong anion exchange resin comprising HCO₃ ⁻ as counterions, quatemaryamine functional groups and the resin is based onpolystyrene-divinylbenzene.

Suitably, water is used to elute the ⁶⁸Ga from the anion exchanger inthe method according to the invention.

EXAMPLES Examples 1 Comparison of ⁶⁸Ga-radiolabelling ofDOTA-D-Phe³-Tyr¹-Octreotide (DOTA-TOC) Using Conventional Heating andMicrowave Activation

1a) ⁶⁸Ga-radiolabelling of DOTA-TOC Using Conventional Heating

Sodium acetate was added to the eluate from a ⁶⁸Ge/⁶⁸Ga-generator (36 mgto 1 mL) to adjust the pH of the eluate to approximately 5.5 and themixture was vortexed well. DOTA-TOC (20 nmol) was added and the reactionmixture was heated at 96° C. for 25 min. The reaction mixture was cooledto room temperature and applied to a C-18 SPE-column (HyperSEP S C18),which was then washed with 2 mL H₂O and the product was eluted withethanol: water 50:50 (1 mL).

The reaction mixture and the product were analysed by HPLC using VydacRP and Fast Desalting HR 10/10 FPLC gel filtration columns.

The analytical radiochemical yield (RCY) was 67%.

The isolated RCY was 34%.

Electrospray ionization mass spectrometry, ESI-MS, was performed onFisons Platform (Micromass, Manchester, UK), using positive modescanning and detecting [M+2H]²⁺. DOTATOC was detected at m/z=711.26 andauthentic Ga-DOTATOC was detected at m/z=746.0 (calculated m/z=746.5).

1b) ⁶⁸Ga-Radiolabelling of DOTA-TOC Using Microwave Activation

The reaction mixture was prepared identically as described under 1a) andtransferred into a Pyrex glass vial for microwave activation for 1 minat 100 W. The reaction mixture was cooled to room temperature andapplied to a C-18 SPE-column (HyperSEP S C18), which was then washedwith 2 mL H₂O and the product was eluted with ethanol:water 50:50 (1mL).

The reaction mixture and the product were analysed by HPLC using VydacRP and Fast Desalting HR 10/10 FPLC gel filtration columns.

The analytical RCY was over 98%.

The isolated RCY was 70%.

Electrospray ionization mass spectrometry, ESI-MS, was performed onFisons Platform (Micromass, Manchester, UK), using positive modescanning and detecting [M+2H]²⁺. DOTATOC was detected at m/z=711.26 andauthentic Ga-DOTATOC was detected at m/z=746.0 (calculated m/z=746.5).

1c) Results of the Comparison

In the case of microwave activation, the amount of radioactive materialand the product specific activity was increased by 21%. The isolatedradiochemical yield was increased 2 fold compared to the resultsobtained with conventional heating. As the radiochemical yield of thereaction mixture in case of microwave activation was over 98%, a furtherpurification would not have been necessary and the crude reactionmixture could have been used for in vivo application.

Example 2 ⁶⁸Ga Radiolabelling of DOTA Linked to Oligonucleotides

In a first step, four different antisense oligonucleotides specific foractivated human K-ras oncogene were linked to DOTA:

-   17-mer phosphodiester oligonucleotide with hexylaminolinker at 5′    end;-   17-mer phosphodiester oligonucleotide with hexylaminolinker at 3′    end;-   17-mer phosphorothioate oligonucleotide with hexylaminolinker at 5′    end; and-   2′-O-methyl phosphodiester with hexylaminolinker at 5′ end.    2a) Conjugation of DOTA to Oligonucleotides:

DOTA (32 mg, 66 μmol) and Sulfo-NHS (14 mg, 65 μmol) in H₂O (250 μl)were added to EDC (13 mg, 68 μmol) in H₂O (250 μl), stirred on ice for30 min and then warmed to room temperature to give DOTA-sulfo-NHS. A 100fold excess of DOTA-NHS solution was added dropwise to theoligonucleotide (70-450 nmol) in 1M carbonate buffer (pH 9) and thencooled on ice. The mixture was left at room temperature for 10 hours.The reaction mixture was first purified by gel filtration with NAP 5columns, eluted with H₂O and 100 μL of 1M TEAA (triethylammonium acetatebuffer) was added to 1 mL of the product eluate. The product eluate wasthen applied to a C-18 SPE column (Supelco), the column was washed with50 mM TEAA (5 mL), 50 mM TEAA containing 5% acetonitrile (3 mL) and theDOTA-oligonucleotide was eluted with water:acetonitrile 50:50 (1 mL).The water-acetonitrile fraction was dried using a vacuum centrifuge. Theproducts were analysed using electrospray ionization mass spectrometry.Analysis in negative mode after direct infusion resulted in thefollowing data: 1. DOTA-phosphodiester: MS (ESI⁻) m/z: 662.27 [M−8H]⁸⁻;756.36 [M−7H]⁷⁻; 882.91 [M−6H]⁶⁻. Reconstitution of the data gaveM=5303.71; 2. DOTA-phosphorotioate: MS (ES⁻) m/z: 656.58 [M−8H]⁹⁻;738.56 [M−7H]⁸⁻. Reconstitution of the data gave M=5917.35; 3.DOTA-2′-O-methyl phosphodiester: MS (ESI⁻) m/z: 674.02 [M−6H]⁹⁻; 770.19[M−8H]⁸⁻; 885.00 [M−7H]⁷⁻. Reconstitution of the data gave M=6148.84.

2b) ⁶⁸Ga-Radiolabelling

Sodium acetate was added to the eluate from a ⁶⁸Ge/⁶⁸Ga-generator (36 mgto 1 ml) to adjust the pH of the eluate to approximately 5.5 and themixture was vortexed well. DOTA-oligonucleotide (10-100 nmol) was addedand the mixture was transferred into a Pyrex glass vial for microwaveactivation for 1 min at 100 W. The reaction mixture was cooled to roomtemperature then 1 mL of 150 mM TEAA in H₂O was added. The mixture wasapplied to a C-18 SPE-column (Supelco), which was then washed with 50 mMTEAA (1 mL), 50 mM TEAA containing 5% acetonitrile (1 mL). The productwas eluted with ethanol: water 50:50 (1 mL) or water:acetonitrile 50:50(1 mL). The reaction mixture was analysed by HPLC using Vydac RP andFast Desalting HR 10/10 FPLC gel filtration columns. The analytical RCYranged from 50% to 70%, the isolated RCY ranged from 30 to 52%. Largeramounts of stronger eluents might improve the isolated RCY.

Example 3 ⁶⁸Ga Radiolabelling of DOTA Linked to Peptides

In a first step, four different peptides were linked to DOTA:

-   Vasoactive Intestinal Peptide (VIP); 28 amino acid residues;-   Neuropeptide Y Fragment 18-36 (NPY); 19 amino acid residues;-   Pancreastatin Fragment 37-52 (P); 16 amino acid residues; and-   Angiotensin II (A); 8 amino acid residues.    3a) Conjugation of DOTA to Peptides:

Conjugation was carried out as described in 2a) using peptides (0.5-3μmol) instead of oligonucleotides.

The reaction mixtures and products were analysed by HPLC using Vydac RPand Fast Desalting HR 10/10 FPLC gel filtration columns. Electrosprayionization mass spectrometry, ESI-MS, was performed on Fisons Platform(Micromass, Manchester, UK), using positive mode scanning and detecting[M+2H]²⁺, [M+4H]⁴⁺ and [M+5H]⁵⁺. VIP was detected at m/z=832.07[M+4H]⁴⁺. (DOTA)₂-VIP was detected at m/z=1025.00 [M+4H]⁴⁺. (DOTA)₃-VIPwas detected at m/z=1122.0 [M+4H]⁴⁺, (DOTA)₄-VIP was detected atm/z=1218.00 [M+4H]⁴⁺. NPY was detected at m/z=819.31 [M+3H]³⁺. DOTA-NPYwas detected at m/z=948.18 [M+3H]³⁺. P was detected at m/z=909.55[M+2H]²⁺. DOTA-P was detected at m/z=1103.02 [M+2H]²⁺. A was detected atm/z=524.1 [M+2H]²⁺ and DOTA-A was detected at m/z=717.20 [M+2H]²⁺.

3b) ⁶⁸Ga-Radiolabelling

⁶⁸Ga-radiolabelling was carried out as described in 2b) using 10-20 nmolDOTA-peptide.

The reaction mixture was analysed by HPLC using Vydac RP and FastDesalting HR 10/10 FPLC gel filtration columns. The analytical RCYranged from 80% to 90%, the isolated RCY ranged from 60 to 70%. Largeramounts of stronger eluents might improve the isolated RCY.

1. A method of producing a radiolabelled gallium complex in a formsuitable for use in PET or SPECT radiopharmaceutical imaging, saidmethod comprising reacting a Ga³⁺ radioisotope in a suitable solventwith a macrocyclic bifunctional chelating agent, wherein saidmacrocyclic bifunctional chelating agent is linked to a targeting vectorselected from the group consisting of proteins, glycoproteins,lipoproteins, polypeptides, glycopolypeptides, lipopolypeptides,peptides, glycopeptides, lipopeptides, carbohydrates, nucleic acids,oligonucleotides or small organic molecules; characterised in that thereaction is carried out using microwave activation at 80 to 120 W for 20s to 2 min.
 2. The method according to claim 1 wherein the Ga³⁺radioisotope is selected from the group consisting of ⁶⁶Ga³⁺, ⁶⁷Ga³⁺ and⁶⁸Ga³⁺.
 3. The method according to claim 1 wherein the Ga³⁺ radioisotopeis ⁶⁸Ga³⁺.
 4. The method according to claim 1 wherein the macrocyclicbifunctional chelating agent comprises hard donor atoms, preferably Oand N atoms.
 5. The method according to claim 1 wherein the targetvector is a peptide or oligonucleotide.
 6. The method according to claim1 wherein the microwave activation is carried out at 90 to 110 W.
 7. Themethod according to claim 1 wherein the microwave activation is carriedout for 30 s to 90 s.
 8. The method according to claim 3 wherein the⁶⁸Ga³⁺ is obtained by contacting the eluate from a ⁶⁸Ge/⁶⁸Ga generatorwith an anion exchanger and eluting ⁶⁸Ga³⁺ from said anion exchanger. 9.The method according to claim 8 wherein the ⁶⁸Ge/⁶⁸Ga generatorcomprises a column comprising titanium dioxide.
 10. The method accordingto claim 8 wherein the anion exchanger comprises HCO₃ ⁻ as counterions.11. The method according to claim 8 wherein the anion exchanger is ananion exchanger comprising quaternary amine functional groups, or theion exchanger is a anion exchange resin based onpolystyrene-divinylbenzene.
 12. The method according to claim 1 for theproduction of ⁶⁸Ga-radiolabelled PET tracers.
 13. Method according toclaim 8 wherein the eluting ⁶⁸Ga³⁺ is in the picomolar to nanomolarrange after the elution, and more preferably in a nanomolar tomicromolar level.